GB2111829A

GB2111829A - A process for the preparation of lyophilized vaccine against duck hepatitis

Info

Publication number: GB2111829A
Application number: GB08236291A
Authority: GB
Inventors: Dr Erzsebet Horvath; Gabor Tollas
Original assignee: PHYLAXIA OLTOANYAGTERMELOE
Current assignee: PHYLAXIA OLTOANYAGTERMELOE
Priority date: 1981-12-23
Filing date: 1982-12-21
Publication date: 1983-07-13
Also published as: PL133923B1; NL8204915A; PL239715A1; HU183765B; US4622222A; SU1237081A3; GB2111829B

Description

1 GB 2 111829 A 1

SPECIFICATION

A process for the preparation of a Iyophilized vaccine against duck virus hepatitis The invention relates to a process for the preparation of a Iyophi 1 ized vacci ne against duck hepatitis by using 5 the attenuated hepatitis virus TN cultivated by Asplin.

Duck hepatitis is a dangerous viral disease to which ducklings are susceptible in the first 4-5 weeks of their life. If they are not protected against this disease, 40 to 50% of the young animals can perish. Therefore a protection method was sought and a vaccine suitable for immunization was produced. For the preparation of the presently used vaccine the attenuated TN virus cultivated byAsplin is used (Asplin F.D. and McLauchian 10 J.D.: Vet.Rec. 1954 32, 66,456; Asplin F.D.: Vet. Rec. 1956, 68,412). The virus, the titre of which has to be at least 104 E1D50/mi, is injected into the allantoic cavity of 1 0- day embryonated hen's egg in a dilution between 1: 10 and 1: 20, the eggs are candied daily and the eggs containing dead embryos are taken out and eliminated for the first 24 hours. Eggs are suitable for the preparation of the vaccine when the embryo dies between 24 and 96 hours after the inoculation. The allantoamniotic fluid of the eggs (that is the fluid surrounding the embryo) is drawn off by vacuum, treated with antibiotics and filled into bottles.

The process was doubtlessly a great advance in the field of intensive large-scale poultry breeding; the vaccine, owever, possesses disadvantages, too. The main disadvantage is that it is fluid and has to be stored in frozen state at a temperature of at least -20'C. The substance can be stored in this state for at most half a year. In the course of the transport of the frozen vaccine breakings often occur, and therefore it is preferred to transport it personally to the destination. After melting (calculated from the time of the delivery) it can be stored at a temperature of +40C for 7 days. The vaccine is suitable for use if its titre reaches the value of 1 O"Ell)50/M1. According to our own examinations this vaccine cannot be Iyophilized, although tests were carried out with numerous protective materials. The virus loss caused by the Iyophilization is too high, and the biological activity of the Iyophilized substance is not sufficient.

In the production of this vaccine the material consumption is very high; for the preparation of 100 litres of vaccine 23,000 embryonated hen's eggs are necessary.

The aim of the invention was to develop a process for producing a vaccine of high titre by using less raw material, which is Iyophilizable and storable for a longer time and - due to its dry state - easier to handle.

It is known that the quantity of virus accruing in embryonated hen's egg is not the same in all parts of the 30 egg (Hwang J. and Dougherty E.: Avian Dis. 1964, 19 0,264). The virus accrues mainly in the body of the chicken embryo, particularly in the liver, while the allantoamniotic fluid - the material from which the traditional vaccine is isolated - contains the least virus.

In order to solve the above task a process for the preparation of a Iyophilized vaccine against duck hepatitis was developed by using the attenuated hepatitis virus TN cultivated by Asplin. According to the invention the virus deposited in the Strain Collection of the Hungarian Institute of Public Health on 27/11181 under No. 00220 is injected into the allantoic cavity of 10-day embryonated SPF- Inen's eggs; the eggs are incubated at a temperature of about 370C; the embryos dying between the 24 and 96 hours after the inoculation are collected; the harder parts of them are optionally eliminated under sterile conditions; then the embryos are homogenized with physiological saline solution, centrifuged, and antibiotics are added to the pure suspension; and after the addition of protective and skeleton forming (carrier or matrix-forming) agents the virus substance is freeze-dried by well-known methods.

The SPF (specific pathogen free) eggs for the present process must be free from Avian encephalomyelitis, fowl plague, EDS, bronchitis, leucosis A-13, Gumboro, Marek, Reovirus as well as from Salmonella Gall., Salmonella typhi murium, Mycoplasma Gall., Mycoplasma Synoviae and the antibodies of these, respectively.

Under homogenization the suspension is advantageously diluted to such an extent that the substance consists of 25 to 35 % by volume of embryo and 75 to 65 % by volume of physiological saline solution.

It is particularly advantageous to use a combination of polyvinyl pyrrolidone, gelatine, glucose and saccharose as protective and skeleton forming agent.

Lyophilization is preferably effected as follows: the shelves of the freeze-drying machine are precooled to a temperature between 0 and -6'C; the substance put on the shelves is cooled to a temperature between -30 and -40 OC; the water in the substance is sublimated in vacuum; after the sublimation the shelves are heated to a temperature between +30 and +40'C and thus the substance is dried for 4 to 10 hours.

When performing the process of the invention one proceeds so that the virus is inoculated in diluted state into the allantoic cavity of the 1 0day embryonated SPF-egg in a dose of 10,000 E1D50 per egg. After the inoculation the embryos dying within the first 24 hours are eliminated. The eggs are candied daily, collected in the 48th, 72nd and 96th hour, and eggs containing the dead embryos are stored until use at a temperature of +4C. The front part of the embryo's head (eyes, bill) is removed under sterile conditions, the bill because it is hard and the eyes because they discolour the vaccine.

The embryos are smashed in a mixer, then they are further homogenized in an ultra-homogenizer (e.g. in an ultraturrax machine) while physiological saline solution is added. The suspension contains about 50 to 80 % by volume of physiological saline solution. The suspension is centrifuged at a speed of rotation of 2000-3000 minutes-' for 20 minutes, and the cleared middle part is filtered through four layers of gauze while the bone residues on the bottom of the centrifuge glass and the feather fundaments floating on the top 65 2 GB 2 111829 A 2 of the substance are removed. In orderto examinethe purity of the filtered substance it is spread on culture-media and treated with antibiotics (penicillin, streptomycin, neomycin and chloramphenicol). If the substance proves to be infected after a certain time, gentamycin is added. The sterile substance is Iyophilized within 5-6 days from harvesting; in the meantime it is stored at +4'C.

Before the Iyophilization skeleton forming and protective agents are added to the virus suspension. The 5 following combination is advantageous:

5% of collidone solution (polyvinylpyrrolidone, 10% by volume) 5% of gelatine solution (10 % by volume) 4% of glucose solution (50 % by volume) 3 % of saccharose solution (50 % by volume).

The Iyophilization can be carried out by well-known methods. It will be described in the examples in detail. The inoculum strain virus is prepared similarly to the vaccine but here the sterility has to be maximised, because no antibiotic can be added to the inoculum virus.

The Iyophilized vaccine can be stored at a temperature of +4'C for one year.

Considering the fact that the titre of the virus suspension after Iyophilization is at least one magnitude higher than that of the vaccine which has to be stored in frozen state, the quantity to be pepared is reduced to a tenth. Due to this fact much fewer eggs are used, and the price of the embryonated eggs amounts to 95 % 20 of the production cost. At the same time a significant saving of labour is attained.

Depending on the virus titre, by diluting a 2 mi via[ 100 to 1000 single doses can be obtained, according to the following relation:

titre 105 E1D50/M1 100 19 Number of Necessary quantity of buffer doses for the dilution ducklings M1 105.3 E1D5o/mi 200 105.7 ElDro/ml 500 m] mI 106 E050/M1 1000 100 mI laying ducks mi mi 500 ml 1000 mi The dose in case of both the duckling and the laying duck is at least 2000 E1D50that meansthat dueto the different dilution 0.1 mi of vaccine in the case of duckling and 1 mi of vaccine in the case of laying duck is added. The vaccine can be administered by injection or - in the case of ducklings - in drinking water. It is recommended to inoculate the breeding stock twice before the laying period. The laying ducks pass the immunityto the ducklings through the yolk but the thus-obtained protection lasts only for about 14 days; therefore the ducklings, too, have to be treated. If the vaccine is added to the drinking water, it is recommended to have the stock thirsted for some hours and to give the vaccinic water only after it. By moving the animals it can be ensured that every duckling can drink and drinking vessels should be used in which the ducklings cannot bathe.

The invention is illustrated in detail by the following examples.

EXAMPLE 1

Preparation andlyophilization of the vaccine 10-day embryonated hen's eggs of SPF quantity are candled,the limit of the air-sack and the place of the embryo are marked on the shell, and the blunt end of the egg is disinfected. The suspension of the egg-inoculation virus prepared with sterile physiological saline solution is injected into the allantoic cavity through a hole made above the airsack. 0.2 m] of the substance containing approximately 50,000 E1D50 virus 55 quantity per millilitre is used for each egg. Then the hole is closed with paraffin and the eggs are incubated in hatchers at 370C. The embryos dying within the first 24 hours as well as those still living after 96 hours are eliminated. The eggs are candied daily and the eggs containing dead embryos are stored between +2'C and +WC until use.

Under sterile conditions, the embryos are collected, the front part of the head is cut off with a pair of scissors, then the embryos are crushed in a mixer and diluted with physiological saline solution, and then then crushed again in an ultraturrax homogenizer. The embryo squash is further diluted and the obtained suspension contains 70% of phyisological saline solution. This suspension is centrifuged at a speed of rotation of 3000 minutes-' for 20 minutes and the clear middle part is filtered through four layers of sterile gauze.

3 GB 2 111 829 A 3 The thus-obtained material is submitted to a purity examination; then directly afterthe examination it is treated with antibiotics by using 100, 000 IU of penicillin, 760,000 IU of streptomycin, 0.2 g of chloramphenicol and 0.5 g of neomicyn for one litre of suspension. The treated suspension is allowed to stand at room temperature for one hour, and then inoculated on culture media which is suitable for cultivation of aerobic and anaerobic bacteria as well as fungi. If within 3 days no growth of a microorganism begins on the culture media kept at room temperature, in a thermostat, the material is considered sterile. If it proves infected, 0.3 g of gentamycin per litre is added to it. The suspension is Iyophilized as soon as possible, but at least within 5-6 days after harvesting. Until that time it is stored at a temperature of + 4'C. Before the Iyophilization the following sterile protective agents are added to the suspension:

% of collidone solution (10 % by volume) 5 % of gelatine solution (10 % by volume) 4 % of glucose solution (50 % by volume) 3 % of saccharose solution (50 % by volume).

is The total quantity of the protective and skeleton forming agents amounts to 17 %. Before the Iyophilization the titre of the suspension is determined 107 E 1 D50/m 1.

The virus suspension is filled in doses of 2 mi into vials and is put onto the material keeping shelves of the freeze-drying machine precooled to a temperature of -5'C. When the temperature of the material reduced to -450C, the vacuum pump is operated. When the pressure decreased to 0.1 mbar, the heating of the material 20 keeping shelves is turned on and adjusted so that the temperature of the material should be between -30 and 35'C (for a pressure of 0.1 mbar a shelf temperature of about - 1 5oC is needed). When the sublimation is finished, the shelves are heated to a temperature of +35'C and the maximum vacuum is put on so the material is desiccated as it is possible. After 8 hours drying time the water content of the product is under a value of 2 %.The vials are stoppered while in the Iyophilizer under vacuum and the rubber stopper is 25 protected by metal stop-cap.

EXAMPLE 2 Preparation of the inoculation virus One proceeds as in Example 1, with the difference that no antibiotic is added to the suspension and the ready material is not Iyophilized but stored in deep-frozen state in liquid nitrogen. Its titre is at least 1 06.5_ 107 E1D50/mi. Before use a dilution is prepared from the inoculation virus in a ratio of 1: 60 to 1: 200; 0.2 mi of which contain about 10,000 E1D50 of the virus. The inoculation virus cannot haemagglutinate the erythrocytes of the hen; EXAMPLE 3 Detection of the immune state The absence of an appropriate virulent virus the immune state was evaluated with the help of a virus neutralization test carried out with serum. The 480 test ducklings came from a farm where the breeding stock 40 was not immunized due to export interests. One group consisted of 20 test animals.

In the vaccination tests PBS buffer of the following composition was used for the dilution:

NaCI 8 g I(C1 0.2 g 45 Na2HP04.2H20 1.15 g KH2P04 0.29 50 CaC12 0.1 g M9C12.6H20 0.1 g distilled water ad 1,000.0 mi 55 Different dilutions were prepared from the vaccine prepared according to Example 1 with the buffer of the above composition, and immunization tests were carried out by injecting the dilutions subcutaneously or added the vaccine in water. For comparison tests were carried out with the traditional liquid vaccine, too. 60 12 days after the immunization blood samples were taken and the serum was inactivated at 56'C for 30 minutes, and the antibody level was determined with the virus dilution method. The dilution of the serum in a ratio of 1: 10 was mixed with the individual virus dilutions, and the thus-prepared mixtures were allowed to stand at room temperature for 1 hour and then injected into 11 -day embryonated SP17-eggs. The virus titre and the neutralization index (NI), respectively, were determined on the basis of the number of embryos 65 4 GB 2 111 829 A which died or were still living, but showed characteristic pathological alterations after one week and was calculated by Reed-Muench method. The results are summarized in the following table:

4 Method of Dose Type of NI Difference treatment E1D50 administra- re I ative to 5 tion the control in% control 1.6 10 liquid vaccine 50 subcut. 2 25 613 subcut. 3 87.5 6130 subcut. 3.6 125 Iyophilized vaccine according to the invention 158 subcut. 2.6 62.5 1585 subcut.

4.6 4187.5 15850 subcut. 4.8 200 Iyophilized vaccine according to the invention 3160 watering. 3.7 131 31600 watering 4.4 175 316000 watering 4.6 187.5 22 5 An index above 2 is already positive, an index above 3 is considered as a very good result. From the table it 35 comes clear that the protective effect of the vaccine of the invention is excellent.

EXAMPLE4

Proving of the storability of the vaccine The storability of the vaccine was examined by modelling the actual storage as well as by usual thermal 40 load. In modelling the storage one started from the fact that in most countries the vaccines have to be stored at a temperature of +40C according to the regulations; thus the product for export, too, was examined under such conditions. The vaccine stored at a temperature between +2 and +5'C lost 0.6 exponent (from the exponent of EID50) of the activity within 12 months. This is an irrelevant loss, but corresponds to the scattering among the virus contents of the vials.

The thermal load test renders possible a rapid examination. If the titre of the vaccine stored at 370C for 1 week is still acceptable, it can be used for one year when stored according to the regulations. The vaccine prepared according to the invention lost 0.5 exponent of activity when stored at 37'C for 1 week what is a very advantageous result.

Claims

1. A process for the preparation of a Iyophilized vaccine against duck hepatitis by using the attenuated hepatitis virus TN cultivated by Asplin, characterized in that the virus deposited in the Strain Collection of the Hungarian Institute of Public Health under No. 00220 is injected into the allantoic cavity of embryonated SPF-hen's eggs, the eggs are incubated at a temperature of about 37'C, the embryos dying between 24 and 96 hours after the inoculation are collected and their harder parts optionally removed under sterile conditions, the embryos are homogenized with physiological saline solution and centrifuged, antibiotics are added to the pure suspension and after the addition of protective and skeleton forming agent(s) it is Iyophilized.

2. A process as claimed in claim 1 in which a suspension consisting of 25to 35% by volume of embryo and 75 to 65% by volume of said physiological saline solutione is prepared in the homogenisation step.

3. A process as claimed in claim 1 or2 in which a combination of polyvinyl-pyrrolidone, gelatine, glucose and saccharose is used as protective and skeleton forming agent.

4. A process as claimed in anyone of the preceding claims in which in the lyophilization the shelves of 65 GB 2 111 829 A 5 the lyophilizer are precooled to a temperature between 0 and -6T, the material put onto the shelves is cooled to a temperature between -30 and - 40T, the water of the material is sublimated under vacuum, after the sublimation the shelves are heated to a temperature between +30 and +40T and the material is dried for 4 to 10 hours.

5. A process as claimed in claim 1 substantially as described herein in Example 1.

6. Lyophilized duck hepatitis vaccine when prepared by a process as claimed in any oneof the preceding claims.

7. Duck hepatitis vaccine prepared from a lyophilized vaccine as claimed in claim 6.

Printed for Her Majesty's Stationery Office, by Croydon Printing Company Limited, Croydon, Surrey, 1983. Published by The Patent Office, 25 Southampton Buildings, London, WC2A lAY, from which copies may be obtained.